(1) Field of the Invention
The present invention generally relates to miRNA vectors. More specifically, the invention is directed to vectors having more than one miR-30 hairpin that can be expressed by RNA polymerase II.
(2) Description of the Related Art
A recent advance in gene silencing methodology is to modify the stem sequence of miR-30 microRNA (miRNA) hairpin to achieve knockdown (i.e., reduction of expression) of artificially targeted genes (Zeng el al., 2002; McManus et al., 2002; Boden et al., 2004; Zhou et al., 2005; Stegmeier et al., 2005; Silva et al., 2005; Dickins et al., 2005). Modified miR-30 can achieve more effective knockdown than previous short-hairpin RNA (shRNA)-based methods (Boden et al., 2004; Silva et al., 2005) and can be expressed from pol II promoters. Nevertheless, knockdown efficiencies vary for different target genes or with different ways of expressing the modified miR-30, especially when knockdown vectors are present in low numbers in target cells (Stegmeier et al., 2005; Silva et al., 2005; Dickins et al., 2005). Inadequate efficiencies of gene knockdown for various research and therapeutic purposes remain frequently encountered.
For microRNA mediated gene silencing, one potential means to improve gene silencing efficiency is to express multiple copies of the microRNA hairpin from a single transcript. This concept originated from the finding that microRNA hairpins are often processed from within larger RNA polymerase II (pol II) transcripts (Kim, 2005). Since primary pol II transcripts can encode multiple genes (polycistronic), it was presumed that multiple hairpins could also be processed out of one pol II transcript. In nature, closely clustered microRNA hairpins have been found in the genome of many species, including man (refs, and figure XX), suggesting that multiple microRNA hairpins could be processed from one transcript. Artificial constructs expressing more than one microRNA hairpin have been made, with variable results (Zhou et al., 2005; Chung et al., 2006; PCT Publication No. WO 2006/044322 A2).